Project Summary Proteolytic disorders represent a spectrum of degenerative conditions of the structural extracellular matrix (ECM; collagen, elastin) resulting from chronic imbalances between proteases & anti-proteases. Unlike collagen, elastic fibers, which enable tissue recoil, poorly auto-regenerate and repair in adult tissues and are a critical ?missing link? in natural reparative responses to tissue injury, and represent an unsurmountable impediment to effecting tissue repair in proteolytic disorders. The goal of this proposal is thus to test the feasibility of a novel ECM regenerative therapy based on the use of drug delivery nanoparticles (NPs) to restore elastic matrix homeostasis in a proteolytic milieu. We will test our approach in an example proteolytic disorder, pelvic organ prolapse (POP), which is characterized by bulging or herniation of one or more pelvic organs into or out of the vagina, and which severely diminishes quality of life of many women. Thre is no treatment for early manifestations of POP to prevent further progression and treatments for later stage POP are primarily surgical and have high complication and revision rates. These factors, and the relatively straightforward delivery of therapeutics to easily accessed vaginal tissues, render POP an apt disease platform to develop and test our approach. Recent evidence including our own studies in a lysyl oxidase-like 1 knockout (LOXL1 KO) mouse model of POP, which evokes human POP pathophysiology, suggest that chronic proteolysis and impaired elastin regenerative repair in the vaginal wall after vaginal delivery, are linked to POP development. In cultures of non-epithelial vaginal cells (NEVCs) from LOXL1 KO mice with late stage POP, we also showed that it is feasible to reverse these aberrations with the novel pro-elastogenic matrix regenerative factors (MRFs) we previously identified. To enable localized, controlled and sustained MRF release within POP tissues in vivo we have developed novel biodegradable polymer NPs, which independent of the MRFs they release, are surface modified with cationic amphiphiles which impart pro-elastogenic & anti-proteolytic properties. Our specific aims (SAs) will test the hypothesis that localized one time vaginal treatment with our cationic amphiphile-modified MRF-NPs in LOXL1 KO mice in early stage POP will augment quantity & quality (matrix yield, crosslinking, fiber density) of elastic matrix towards restoring vaginal ECM homeostasis. SA1 will identify MRF-NP formulations that significantly augment new ECM deposition & anti-proteolytic outcomes in cultures of NEVCs from LOXL1 KO mice with early stage POP. SA2 will investigate MRF-NP safety and ECM regenerative responses in LOXL1 KO mice in early POP. Although new and untested, our approach promises high rewards in arresting or regressing POP development at an early stage, not presently possible. Feasibility data generated in this study will guide follow up long-term preclinical studies in the same model to assess therapeutic effectiveness of our treatment and benefits of repeat NP infusions. Validating this approach will also allow us to extend its application to other proteolytic disorders, with suitable design modifications based on preferred route of delivery to target tissues.